Study Of Correlation Between Infection Of HPV In Esophageal Squamous Cell Carcinoma And Prognosis Of Patients

Esophageal carcinoma is the eighth most common cancer and the sixth most common cause of death from cancer, worldwide. Despite increasing rates of esophageal adenocarcinoma in many western countries, esophageal squamous cell carcinoma (ESCC) remains the dominant histological type of esophageal cancer worldwide. China is located in the famous "Asian esophageal cancer belt", the morbidity and mortality of esophageal cancer in China has been ranked first in the world. Esophageal carcinoma is the fifth most common cancer and the fourth most common cause of death from cancer in China, and more than90%of cases are ESCC. Once developed, esophageal cancer usually rapidly invades surrounding tissues and lymph nodes. Due to the absence of early symptoms, invasiveness of the disease, and its late diagnosis, it is generally associated with a poor prognosis. The etiology of ESCC remains unclear, and epidemiological studies suggest that tobacco smoking, heavy alcohol drinking, micronutrient deficiency, and dietary carcinogen exposure may cause the malignancy. Infectious agents have been implicated, as either direct carcinogens or promoters. In particular, human papillomavirus (HPV) has been postulated as a possible cause of ESCC. HPV is currently one of the most common sexually transmitted infections worldwide. HPV is a small, nonenveloped double-stranded DNA virus with tropism for the squamous epithelium where it can cause hyperproliferative lesions, and then cause carcinogenesis. HPV with potential for oncogenesis based on persistent infection is known as the high risk HPV. The association between infection of HPV and genital cancers, especially cervical cancer has been well established, the role of HPV in nongenital cancers has become the hotspot of researchers in the world. According to previous studies. HPV-16is the most prevalent type in squamous cell carcinoma, followed by HPV-18. while other high-risk HPV types are rare. Increased numbers of vaginal, oral, and oral-anal sex partners have been associated with an increased risk of developing HPV-associated oropharyngeal cancer. Because the histological similarities between the oropharyngeal squamous epithelia and esophagus HPV infection in esophageal cancer was first suggested in1982based on histological observations by Syrjanen. The etiological role of HPV in ESCC is still unclear. Once HPV infection has taken place, viral DNA synthesis occurs. Additional accumulating changes can lead to transformation. These changes are a multistage complex interaction of the host immune system in combination with the expression of two viral oncogenes:E6and E7, followed by a series of epigenetic changes occurring in dysplastic lesions. The viral oncogene products E6and E7play a key role in HPV-associated carcinogenesis, abrogating p53and retinoblastoma tumor suppressor functions, respectively. E7binds to and degrades Rb, releasing E2F, leading to p16INK4a overexpression, hereafter denoted as p16, which is associated with superior clinical outcome. Thus HPV-positive tumors are characterized by high expression of p16and p16is widely considered a surrogate marker for HPV infection in the context of squamous cell carcinoma. Estimates of the prevalence of HPV in esophageal tumors range widely, from real-time polymerase chain reaction assays to type-specific DNA in-situ hybridization. The price of these two kinds of detection method is relatively expensive, high technical requirements, the need for advanced laboratory facilities and experienced technical staff, so restrict their wide detection application. Immunohistochemical technique is operated simply, the price is lower, is not so higher on technical requirements, Can be widely popularized. With the present study, we aim to determine the prevalence of HPV infection in ESCC, determine expression of HPV-associated proteins, and evaluate its clinical significance. We also sought to evaluate the effect of tumor HPV status on survival of patients with ESCC and want to confirm p16immunohistochemistry is a very good surrogate marker of HPV infection for ESCC. The study was divided into two parts.Part1HPV infection in ESCC and its relationship to the prognosis of patientsObjective:Human papillomavirus (HPV) as a risk factor for esophageal squamous cell carcinoma (ESCC) has previously been studied, but importance of HPV status in ESCC for prognosis is less clear.Methods:A total of105patients who underwent esophagectomy for ESCC were included in this study. All specimens were evaluated for HPV-16and HPV-18with using the in situ hybridization catalyzed signal amplification method for biotinylated probes. The5-year overall survival (OS) and progression free survival (PFS) were calculated in relation to HPV status and the Cox proportional hazards model was used to determine the hazard ratio (HR) of variables in univariate and multivariate analysis.Results:One hundred and five patients (84men and21women) were included in this study. The median age of the patients was60(range,42-78) years at the date of surgery. Follow up was complete. HPV was detected in27.6%(29) of the105patients with ESCC, and all positive cases were HPV-16. None were positive for HPV-18DNA. There were no significant differences between the two groups with respect to gender, age, pT status, pN status, TNM stage (AJCC), differentiation grade, adjuvant therapy, smoking, and alcohol consumption and tumor location. The5-year OS rate and PFS rate were65.9%and61.8%, respectively, in the HPV-positive group and43.3%,36.8%, respectively, in the HPV-negative group. The difference of survival curves between the two groups remained statistically significant. In the Cox proportional hazards model analysis of5-year survival, the HR of HPV-positive/negative was0.31(95%CI:0.14-0.68; P=0.004) and0.37(95%CI:0.16-0.82; P=0.01), respectively. In the Cox proportional hazards model analysis of5-year PFS, the HR for HPV-positive/negative was0.33(95%CI:0.16-0.67; P=0.002) and0.38(95%CI:0.18-0.77; P=0.008), respectively. HPV infected patients had better5-year rates of OS and PFS then HPV-negative group (P=0.004and P=0.002by the Log-rank test, respectively). After adjustment for pT status, pN status, TNM stage, and smoking, tumor HPV status was also statistically significantly associated with survival among patients with ESCC.Conclusion:HPV infection may be one of many factors contributing to the development of ESCC and tumor HPV status is an independent prognostic factor for survival among patients with ESCC.Part2Study of correlation between p16INK4a and HPV infection in ESCCObjective:HPV-positive tumors are characterized by high expression of pl6and p16is widely considered a surrogate marker for HPV infection in the context of squamous cell carcinoma. This section is intended to explore the feasibility of p16immunohistochemical staining as an alternative molecular marker of HPV infection in ESCC.Method:A total of105patients who underwent esophagectomy for ESCC were included in this study. All specimens were tested for p16expression by immunohistochemistry. All slides were reviewed by a pathologist specializing in gastrointestinal pathology. Kappa values were calculated with the use of Cohen Kappa test. Using p16expression as a stratification factor, we calculated5-year OS and PFS through the Kaplan-Meier method and Log-rank test. Results:One hundred and five patients (84men and21women) were included in this study. The median age of the patients was60(range,42-78) years at the date of surgery. The median follow-up time was42.4(4-61) months of patients. Follow up was complete. P16was detected in37.1%(39) of the105patients with ESCC. There were no significant differences between p16-positive group and-negative with respect to gender, age, pT status, pN status, TNM stage (AJCC), differentiation grade, adjuvant therapy, smoking, and alcohol consumption and tumor location. P16was detected in86.2%(25) of the29patients who were HPV-positive, and p16was detected in18.4%(14) of the76patients who were HPV-negative (P<0.001). Cohen’s kappa coefficient revealed an agreement in two researchers (kappa=0.61). The5-year OS rate and PFS rate were64.1%and58.7%, respectively, in the p16-positive group and45.5%,37.9%, respectively, in the p16-negative group. The difference of survival curves between the two groups remained statistically significant. P16-positive patients had better5-year rates of OS and PFS then p16-negative group (P=0.02and P=0.007by the Log-rank test, respectively). Using p16expression as a stratification factor, we found differences in OS and PFS that were consistent with those based on HPV status.Conclusion:HPV-positive ESCC tumors were characterized by overexpression of p16protein, p16immunohistochemistry is a very good surrogate marker of HPV infection for ESCC.Conclusion of the study:HPV infection may play an important role in the development of ESCC. P16protein immunohistochemistry is a surrogate marker of HPV infection for ESCC. HPV status in tumor is an independent prognostic factor for survival among patients with ESCC. HPV infection may become a new therapeutic target in ESCC.